## Request for a Temporary Exemption from the Requirement of a Tolerance Aspergillus flavus strain s TC16F, TC35C, TC38B and TC46G in or

on Corn

EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER

EPA has received a pesticide petition from Interregional Research Project Number 4 (IR

- 4), Rutgers University

, 500 College Road East, Suite 201W, Princeton , NJ 08 540 ( on behalf of the Texas Corn Producers Board

, 4205 N orth Interst ate 27, Lubbock, T X 79403 ), proposing

, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a( d), to amend 40 CFR part 180 to establish a temporary tolerance exemption for the microbial pesticide s Aspergillus flavus strain s TC16F, TC35C, TC38B and TC46G in or on c orn

. The purpose of this petition is to establish a temporary tolerance exemption assoc iated with an experimental use p ermit.

Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended, IR

- 4 ( on behalf o f the Texas Corn Producers Board ) has submitted the following summary of information, data, and arguments in support of the pesticide petition. This summary was prepared by IR

- 4 ( on behalf of the Texas Corn Producers Board ), and EPA has not fully evaluated the merits of the pesticide petition. The summary may have been edited by EPA if the terminology used was unclear, the summary contained extraneous material, or the summary unintentionally made the reader conclude that the findings reflected EPA’s positio n and not the position of the petitioner.

I

. IR

- 4 ( on behalf of the Texas Corn Producers Board ) Petition Summary

A. Product N ame and Proposed Use Practices

A

. flavus strain s TC16F, TC35C, TC38B and TC46G , non

- aflatoxin

- producing strain s of A

. fl avus , are pro posed for application to c orn to re duce the incidence of aflatoxin

- producing strains of A. flavus and thereby reduce aflat oxin contamination of c orn

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G

, which do not produce a flatoxin, competitivel y exclude aflatoxin

- producing A. flavus strains without increasing A. flavus in the environment in the long term. A sterile carrier colonized with A

. flavus strai ns TC16F, TC35C, TC38B and TC46G is applied at 10 pounds of end

- use product per acre (total am ount of active ingredient s less than 0.01 lb/acre) . The current submission proposes to establish a temporary exemption from tolerance for residues of A. flavus strain s TC16F, TC35C, TC38B and TC46G in or on c orn

. The experimental use p ermit is for up to 20 ,000 acres of corn in Texas, Oklahoma, Arkansas and Louisiana.2

B. Product Identity/Chemistry

1. Identity of the pesticide and corresponding residues

. The pesticide and corresponding residues are identified as A

. flavus strain s TC16F, TC35C, TC38B a nd TC46G

. All four active ingredients are included on a carrier on the end

- use product FourSure.

2. Magnitude of residue at the time of harvest and method used to determine the residue

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G are naturally occ urring fungi that were originally isolated from cottonseed produced in Texas

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G have been shown to be naturally and consistently associated with c orn

. A. flavus is a widespread fungus. It is particularly well a dapted to the hot regions of the U.S. where it is widespread in the environment. The communities of A. flavus in the desert and in agricultural fields are natur ally composed of both aflatoxin

- producing (toxigenic) and aflatoxin non

producing (atoxigenic) s trains. Both atoxigenic and toxigenic strains have been found on essentially all plant material and soils in the s outhwestern U.S. The goal of applications is to increase the percent of the A

. flavus community composed of the strain s TC16F, TC35C, TC38B

an d TC46G and to decrease the percent of A

. flavus that produce s aflatoxin on the crop and the field.

3. A statement of why an analytical method for detecting and measuring the levels of the pesticide residue are not needed

. A temporary exemption from th e requirement of a tolerance for residues of the microbial pesticide s A

. flavus strains TC16F, TC35C, TC38B and TC46G is being proposed for c orn. All lots of the active ingredient s ( A. flavus strains TC16F, TC35C, TC38B and TC46G ) and the formulated produc t are monitored as part of a rigorous quality control program. Starter cultures of A. flavus used in the production of the end

- use product are always screened for aflatoxin production using thin layer chromatography ( TLC ) and appropriate standards. Quality control standards are zero tolerance in the starter cultures and in the formulated p roduct for aflatoxin production and for A. flavus not identified as A. flavus strains TC16F, TC35C, TC38B or TC46G

. A. flavus strains T C16F, TC35C, TC38B and TC46G have

ne ver been found to produce aflatoxin. Starter cultures of A. flavus strains TC16F, TC35C, TC38B and TC46G as well as the end

- use product containing these active ingredient s are also identified to strain by veg etative compatibility analysis.

C. Mammalian T oxicological Profile

There are existing registrations on other atoxigenic strains of A. flavus

, including AF36 and NRRL 21822

, and it is reasonable to expect these new native strains would be similar to the existing registered products. An acute oral tox icity test was performed whereby a single oral dose of 5,000 milligrams/kilogram (mg/kg) per animal of A

. flavus strain AF36 colonized carrier was adm inistered by gavage to five male and five female Sprague Dawley rats. The oral LD 50 was determined to be g reater than 5,000 mg/kg rat body weight. No clinical si gns were observed during the 14

- day study

, and no abnormalities or adverse effects were observed in any of the rats
upon necropsy.3

An initial pulmonary rat study resulted in lethality in a significan t number of animals treated with either the live A

. flavus strain AF36 with another ingredient or heat killed A

. flavus strain AF36 with another ingredient . Onset of symptoms was rapid after dosing with all deaths occurring by day four of the study. All ra ts surviving to day four of the study recovered

, and all rats sacrificed (as scheduled) on day 8 or day 15 of the study had totally eliminated viable A

. flavus strain AF36 from the lungs, caecal contents, and feces . There was no evidence of infectivity. Th e aetiology of deaths was unclear. It appeared that A

. flavus strain AF36 prepared using the other ingredient caused a severe acute inflammatory response. Retrospective literature review and consultation with a toxicologist supported the theory that the re sponses were a result of a synergism with the other ingredient and/or of the other ingredient’s breakdown products formed during preparation of the spore suspension.

A second rat study was therefore undertaken. In the second study

, the conidia were both w ashed from the carrier a nd suspended in a different ingredient than in the first study . Animals (2 male and 2 female for each treatment level) were dosed at 0, 10 5 , 10 6 , 10 7 , and 10 8 colony

- forming units per rat. There were no clinical signs in any of the treatment groups considered to be associated with the test substance. Rats were sacrificed at day 8 without treatment

- associated mortality. No abnormalities were observed in any of the animals at the macroscopic examination at termination.

Based on these two mammalian studies, it was concluded that A

. flavus strain AF36 does not present either a toxicological or an infectious risk to mammals.

Data waivers were requested for the toxicology studies listed below on the following base s:

( i ) R esearchers and other workers have worked with A

. flavus strain s at the Southern Regional Resear ch Center for over 10 years, in commercial fields since 1996 , and in hand

- picked field plots (1989 to 1994) without report of any adverse health effects;

(ii ) A

. flavus s train s TC16F, TC35C, TC38B and TC46G are widely distributed in the environment

, and their occurrence is natural; and

( iii ) T he label will require applicators and other handlers to wear Personal Protective E quipment (PPE) such as waterproof gloves, a dust /mist filtering respirator with the appropriate NIOSH approval prefix N

- 95, P

- 95, or R

- 95, coveralls, long sl eeved shirt and long pants, shoes, socks, and goggles; therefore, exposure should not be a problem. Applications will involve aerial or ground appl ication by mixers/handlers who are licensed and trained to even handle restricted materials. At the 10 lb/acre application rate of the formulated material, the total amount of active ingredient s is less than 0.01 lb/acre. Applications of A

. flavus strain s TC16F, TC35C, TC38B and TC46G do not significantly impact the total amount of A

. flavus in the soil or crop, but only change the proportion of the atoxigenic A. flavus in relation to the overall soil or crop population. Since the product is applied as a gr anular formulation on a carrier , exposure from drift is minimal.4

1. Acute dermal toxicity

. This study was requested to be waived based upon the lack of toxicity in animals dosed orally with A. flavus strain AF36 . While A

. flavus has been reported to be a dermal sensitizer, this testing is not warranted, since the PPE required on the label will provide adequate protection from dermal exposure to workers and pesticide handlers.

2. Primary dermal irritation

. This study was requested to be waived based upon the lack of toxicity in animals dosed orally with A. flavus strain AF36 . Aerial or ground applications will be made by

m ixers/handlers and applicators who are re quired to wear appropriate PPE and are licensed and trained to even handle r estricted materials. At the 10 lb/acre application rate of the formulated material, the total amount of active ingredient is less than 0.01 lb/acre. Applications do not significantly impact the total amount of A

. flavus in the soil or crop, but only change the proportion of the atoxigenic strain in relation to the overall population. Since the product is applied as a granular formulation on a carrier , exposure from drift is minimal.

3

. Primary eye irritation

. This study was requested to be waived based upon the use of appropriate PPE, including goggles, and other factors listed above. Since the product is applied by aerial or ground equipment as a granular formulation on a carrier , exposure from drift is expected to be minimal.

4. Acute i ntraperitonea l

. This study was requested to be waived based upon the lack of toxicity in animals dosed orally and by intratracheal instillation with A. flavus strain AF36 . In addition, the factors listed above will mitigate exposure, which is expected to be minimal.

5. Genotoxicity, reproductive and developmental toxicity, subchronic toxicity and chronic toxicity testing were not performed, since no adverse effects were anticipated in the acute toxicology Tier 1 studies. Tier II studies are only required if triggere d by adverse effects observed in Tier I studies.

D. Aggregate Exposure

1. Dietary exposure

.

i. Food

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G are naturally occurring organism s that do not produce aflatoxin and are thus safer than A. flavu s that produce s aflatoxin. Proposed uses and application rates will not result in increases in the total population of A. flavus on the mature crop beyond naturally occurring background levels

. A

. flavus strai ns TC16F, TC35C, TC38B and TC46G

, when applied to the soil just prior to tasseling , have been shown to significantly reduce the le vels of aflatoxin in c orn at harvest without changing the quantity of A. flavus on the crop. Furthermore, the proposed use s and application rate s will not increase
exposure of humans to A

. flavus by dietary means . A pplications of A

. flavus strain s TC16F, TC35C, TC38B and TC46G do not increase the indigenous populations of A. flavus associated with the harvested crop. The applications merely alter the composition of the fungal community associated with the mature crop , so that aflatoxin

- producing strains are far less frequent. The result is a mu ch lower incidence of aflatoxin in the crop and in the environment associated with5

the developing and mature crop.

ii

. Drinking wat er

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G are naturally occurring organism s tha t are already wide spread in the environment and are not considered to be a risk to drinking water. Both percolation through soil and municipal treatment of drinking w ater would reduce the possibility of exposure of A. flavus through the drinking water. Applications of A

. flavus strain s TC16F, TC35C, TC38B and TC46G do not in crease the long

- term population of A. flavus in the environment, and thus are not expected to in fluence the relationship of A. flavus to water sources. Applications merely change the composition of the A. flavus community, so that aflatoxin are less common in the environment.

2. Non

- dietary exposure

. The potential for non

- occupational, non

- diet ary exposure to the general population is not expected to be significant and is not expected to present any risk of adverse health effects.

E. Cumulative Exposure

There are existing registrations on other atoxigenic strains of A. flavus

, including AF3 6 and NRRL 21822

, and it is reasonable to expect these new native strains would be similar to the existing registered products whose sole mode of action is to reduce aflatoxin. There are no other registered products containing A

. flavus strain s TC16F, TC35 C, TC38B or TC46G

, and no reasonable concern that there would be any cumulative effects since the active ingredients do not have any toxic endpoints. Data submitted show that the fungal metabolite of concern

, which is aflatoxin

, is not produced by A

. flavu s strain s TC16F, TC35C, TC3 8B or TC46G in the crop or in artificial media in the lab. A

. flavus strain s TC16F, TC35C, TC38B and TC46G have been shown to exclude aflatoxin

- producing fungi competitively from the developing crop and to reduce aflatoxin contam ination of corn . Data show that the proposed use will not result in appreciable increases in the long

- term population of A. flavus on the crop beyond naturally occurring levels. Furthermore, there is no expectation of cumulative effects with other pesticid es.

F. Safety Determination

1. U.S. population

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G are naturally occurring organism s. These strains have low toxicity as demonstrated by the acute oral toxicity study in rats by A. flavus strain AF36

. A. flavus is ubiquitous throughout the hot desert valleys in Arizona. Studies have shown that treatment of co rn fields just prior to tasseling with a sterile carrier colonized by A

. flavus strain AF36 at 10 pounds per acre does not increase the long

- te rm pop ulation of A. flavus either on the crop at maturity or in the soil

. Based on this information on A

. flavus strain AF36 , IR

- 4 is of the opinion that the aggregate exposure to A. flavus over a lifetime should not change with application of A

. flavus strain s T C16F, TC35C, TC38B and TC46G

, and exposure to both aflatoxin

- producing A. flavus strains and aflatoxin should decrease. This should be beneficial to human health. Thus, there is a reasonable certainty that no harm will result from aggregate exposure to A

. flavus strain s TC16F, TC35C, TC38B and TC46G

.6

2. Infants and children

. Based on natural occurrence and the lack of toxicity , there is reasonable certainty that no harm to infants, children, or adults will result from aggregate exposure to A

. flavus s train s TC16F, TC35C, TC38B and TC46G . Exempting A

. flavus strain s TC16F, TC35C, TC38B and TC46G from the requirement of a tolerance should pose no significant risk to humans.

G. Effects on the Immune and Endocrine Systems

A

. flavus strain s TC16F, TC35C , TC38B and TC46G are naturally occurring organism s that do
not produce aflatoxin and are thus safer than A. flavus that produce s aflatoxin. To date

, there is no evidence to suggest that A

. flavus strain s TC16F, TC35C, TC38B and TC46G function in a

mann er similar to any known hormone or that they act as endocrine disrupter s

.
H. Existing Tolerances

The re is no existing t olerance exemption for A

. flavus strain s TC16F, TC35C, TC38B and TC46G

. There are permanent tolerance exemption s for A

. flavus strain AF 36 on cotton , corn and pistachio (40 CFR § 180.1206 ) and A. flavus strain NRRL 21882 on peanut and corn (40 CFR § 180.1254).

I. Efficacy Data

The following information was presented for a tolerance exemption for A. flavus strain AF36 on
cotton , corn an d pistachio . The information is relevant for A

. flavus strain s TC16F, TC35C, TC38B and TC46G and for this request for a temporary exemption from the requirement of a tolerance in or

on c orn , as part of a n experimental use p ermit. Existence of aflatoxin in the environment is a public health hazard; as such

, data were submitted to demonstrate that proper use of A

. flavus strain AF36 results in redu ctions in the average aflatoxin

- producing potential of fungi resident in treated areas and in reductio ns in the q uantity of aflatoxin in crops. In field tests with A. flavus strain AF36

, the aflatoxin content of cottonseed was shown to be inversely related to the proportion of the A. flavus community on the crop composed of A

. flavus strain AF36

. Detailed analyses of the aflatoxin content of commercial fields from 1996 through 1998 confirmed that reduced aflatoxin levels were associated with displacement of aflatoxin producers from treated crops and that treatments were associated with up to 90% reductions in crop afl atoxin content. Efficacy of applications of A

. flavus strain AF36 was demonstrated for fungal communities both on cottonseed and corn from treated crops at harvest and in soils of treated fields one year after treatment. This included cotton crops treated in 1996 (112 acres treated), 1997 (463 acres treated), 1998 (499 acres treated ), 1999 (10,488

acres treated ), 2000 (16,725 acres treated ) and 2001 (19,975 acres treated). Displacement of aflatoxin producers is indicated by the proportion of A. flavus commu nities composed of A

. flavus strain AF36. In 1996

, average incidence of A. flavus strain AF36 on treated crops was 88.5% and in th e soil one year after treatment 85.2%. Incidences of A. flavus strain AF36 on treated crops were 78% and 67% in 1997 and 1998 , respectively, and in the soil one year after treatment incidences were 72%7

and 77%, respectively. Successful displacement was also observed as the acreage treated rapidly expanded from 1999 to 2001 with average incidences of A. flavus strain AF36 on trea ted crops ranging from 57% in 1999 to 66% in 2001. Applications were particularly effective a t displacing the high aflatoxin

- producing S strain of A. flavus . S strain isolates produce more aflatoxin than other A. flavus and are prominen t in soils of cotton

- producing areas of California, Arizona and South Texas.

There are preliminary data indi cating that A. flavus strains TC16F, TC35C, TC38B and TC46G (included on a carrier on the end

- use product FourSure) are effective in c orn as well. Efficacies of the four atoxigenic A. flavus strains selected as active ingredients were assessed in laboratory studies on mature kernels. This type of assay reflects increases in toxin that occur in the field when the mature crop receives a rain or when both high humidity a nd high temperature exist after maturation. This type of assay also reflects efficacy that is relevant after the crop has been harvested

, and maize with moisture content above 15% is transported, stored, or even mixed into feed and fed (i.e.

, in the yard a t a dairy). All four atoxigenic strains were highly effective at reducing contamination in these assays

. All four atoxigenic A. flavus strains on a carrier in the end

- use product FourSure demonstrated excellent ability to displace aflatoxin producers and modify the fungal commun ity associated with crops in a field trial . I n three tests over two years (2013 and 2014)

, A. flavus strains TC16F, TC35C, TC38B and TC46G dispersed to the crop and successfully competed with aflatoxin producers. Thus

, the modified A. flavus community was more diverse than when a single atoxigenic genotype is applied as with A. flavus strain AF36 and A. flavus strain NRRL 21882 . There were differences among years, locations , and replicates in which strain was t he most successful. Thi s suggests that slight differences in the crop or environment caused changes in the most successful genotype and further supp orts use of multiple atoxigenic A. flavus strains to provide the best efficacy across the varying crop environments that occur betw een seasons, among locations, and even within individual fields.

The purpose of this temporary exemption from tolerance is to cover the use of A. flavus strains TC16F, TC35C, TC38B and TC46G under a n experimental use p ermit.

J. International Toleran ces

There are no Codex maximum residue levels established for residues of A

. flavus strain s TC16F, TC35C, TC38B and TC46G

. A

. flavus strain s TC16F, TC35C, TC38B and TC46G

- containing products are presently not registered for pest control outside of the Uni ted States.